Distinguishing morphological features of the third larval stage of ovine Trichostrongylus spp.

In this paper we describe a procedure that enables the identification of species of infective third stage (L(3)) Trichostrongylus larvae. Lambs were infected with putatively monospecific infections of three species of Trichostrongylus commonly found in New Zealand (T. axei, T. colubriformis and T. vitrinus) and Teladorsagia (Ostertagia) circumcincta. After recovering L(3) from faecal cultures, the lambs were slaughtered and adult male worms recovered and examined for spicule morphology to verify identification. L(3) were examined for morphological features and measurement of their length. Further L(3) were exsheathed and examined under high power optics to observe posterior morphological features (tubercles). The posterior of T. colubriformis has a three-tubercle structure whereas T. vitrinus has a single tubercle and T. axei none. However, the tails of T. circumcincta also lack tubercles and thus T. axei cannot be readily distinguished from them on this feature. The range of lengths of L(3) of Trichostrongylus spp. (600-858 microm) and T. circumcincta (700-914 microm) were found to overlap considerably. The shape of the anterior end of these two species differs and this in combination with length provides an indication of the proportion of T. axei and T. circumcincta in a culture. A combination of tubercle number, with overall length and anterior morphology of L(3), can be used to differentiate nematode populations of T. axei, T. colubriformis, T. vitrinus and T. circumcincta.

Quantification of total sheep IgE concentration using anti-ovine IgE monoclonal antibodies in an enzyme immunoassay.

Monoclonal antibodies (mAbs) which recognize separate epitopes on ovine immunoglobulin E (IgE) have been used to develop a non-competitive antibody sandwich enzyme immunoassay (EIA) for quantitating ovine IgE. Purified anti-IgE mAb (YD3) coated onto polystyrene microtitre plates was used to capture IgE in serum samples. Biotinylated anti-IgE mAb (XB6) followed by streptavidin conjugated with horseradish peroxidase were used to detect captured IgE. Tetramethylbenzidine and H2O2 were used as enzyme substrate. A reference serum was prepared by pooling sheep sera containing elevated IgE levels. This reference serum was assigned a value of 100 units ml-1 and used to prepare standard curves for the EIA. The linear region of log-log transformed standard curve data covered a range of 0.05-0.8 units ml-1. The equation of a linear regression line fitted to this curve was used to determine sample concentrations. Using purified IgE, 1 unit of reference serum was equivalent to 0.86 micrograms ml-1 IgE. Maximum intra- and inter-assay coefficients of variation for the EIA were 4.6% and 9.7%, respectively. Subjecting serum samples to 15 freeze/thaw cycles, storage at room temperature for 16 days or incubation at 37 degrees C for 8 h resulted in minimal loss of IgE detection. Incubation of serum at 56 degrees C resulted in rapid reduction in detection of IgE by the EIA. The assay was used to determine IgE levels in adult sheep monospecifically infected with weekly doses of the nematode Trichostrongylus axei. Serum IgE levels increased from 9 to 16 days following first infection and reached maximum levels by days 35-58. Serum IgE responses closely followed IgE positive cell responses in the abomasal mucosa.

The PCR typing of MHC-DRB genes in the sheep using primers for an intronic microsatellite: application to nematode parasite resistance.

The strong association between polymorphisms in an intronic microsatellite and the coding sequences for (BoLA)-DRB3 genes, previously described for demonstrating alleles of class II major histocompatibility complex (MHC) in the cow, was examined in sheep to see if similar polymorphisms could be demonstrated in the DRB region of the MHC. The bovine primes LA53 and LA54, previously used to amplify the bovine DRB3 microsatellites, were used with DNA from Australian sheep, eight DRB alleles were identified by length polymorphisms of polymerase chain reaction (PCR) products amplified from the DRB microsatellite region. Incomplete amplification of both alleles was sometimes found for sheep DNA samples using bovine primers, so a modified primer (LA53b) was used, and found to amplify the microsatellite next to intron 2 of the MHC more reliably than the LA53 primer. Two additional primers (LA31 and LA32), used in amplification of the exon 2 region of bovine DRB3, were used in the sheep, and the PCR products were analysed by single-stranded conformation polymorphism (SSCP). These primers successfully amplified the variable region of the ovine DRB region coded by exon 2, and the SSCP technique demonstrated polymorphisms with sheep DNA. Family studies demonstrated the segregation of alleles, by amplification both of intronic microsatellites and of the exon 2 variable region. Close correspondence was found between the two regions for several alleles, suggesting that the intronic microsatellites were closely linked to DRB-variable region alleles. Three families of Merino sheep with different antibody responses to intestinal nematode parasites were examined. The sire group with the highest antibody levels possessed two microsatellite alleles of closely similar length (alleles 3 and 4) inherited from the sire and present in high frequency in the lambs. In contrast, the other two sires did not possess these two alleles and the alleles were in low frequency in their progeny. Further studies are required in unrelated sheep to confirm whether these two alleles are associated with resistance to nematode parasites.

Phenotypic markers for selection of nematode-resistant sheep.

Nematode-resistance of an animal can be defined as an enhanced natural ability, relative to its peers, to both prevent establishment of larval nematodes and evict any that do establish. These parameters are not measurable in a practical sense and consequently nematode-resistance has usually been defined in terms of low faecal nematode egg counts (FEC). Studies in New Zealand and Australia have demonstrated that nematode-resistance, as measured by FEC, has a heritability of about 0.3 in Romney and Merino sheep. However as a selection trait FEC has practical limitations and its use may incur production penalties through withholding drench treatment for prolonged periods or from a need for artificial challenge. FEC is influenced by the level and composition of a natural nematode challenge and especially the expression of the immune response. Thus immunological parameters which reflect the underlying genetic resistance could potentially be used as phenotypic markers. Ideally, a useful phenotypic marker would be easy to sample and its assay would be inexpensive and able to be automated, in addition to being strongly correlated with nematode-resistance. Results from several New Zealand trials have indicated that antibody levels (particularly IgG1) to excretory/secretory antigens of L3 nematodes such as Trichostrongylus colubriformis may meet these criteria. Levels of antibody against L3 antigens are also independent of on-farm drenching strategies. Blood eosinophil count has also been considered for use as a selection parameter but a high degree of sample variability reduces its potential. Other immunological parameters associated with nematode-resistance which have potential as phenotypic markers include serum nematode-specific IgE and products of mucosal mast cells such as proteinases. It is likely that as the critical immune responses of sheep to nematodes become more clearly defined, new immunological parameters with potential for use as phenotypic markers will be found. The definition of these immune responses will also assist in the identification and characterization of genetic markers.

Sequential cellular and humoral responses in the abomasal mucosa and blood of Romney sheep dosed with Trichostrongylus axei.

Abomasal cannulae were surgically placed in 7 2-year-old New Zealand Romney sheep which had been maintained parasite-free from birth. Four of these sheep were randomly selected and dosed orally with 10,000 infective Trichostrongylus axei larvae per week for 8 weeks, while the remaining 3 sheep served as uninfected controls. Abomasal biopsy, blood and faecal samples were obtained from all sheep at regular intervals from 5 days before and until 58 days after the first infection. The sheep were then killed, worm burdens assessed and abomasal and small intestinal samples collected Faecal egg counts of all 4 dosed sheep were low and only one (No. 701) had a substantial worm burden (8400) post mortem. Overall, levels of mucosal mast cells/globule leukocytes, eosinophils, T19+ cells and larval migration inhibitory activity increased significantly in the abomasal mucosa of the dosed sheep compared to the controls. The CD4+:CD8+ cell ratio in the abomasal mucosa of the dosed sheep also increased compared to that of the controls (P = 0.06). In blood, T. axei-specific antibody (total and IgG1) and eosinophil numbers increased significantly in the dosed sheep. Mucosal cells staining for IgE (IgE+), and blood and mucosal eosinophils showed the earliest substantive increases in number followed by increases in specific serum antibody levels, numbers of mucosal cells fluorescing under UV light (UVf) and T19+ cells. The difference in the IgE+ and UVf cell responses indicated that expansion of globule leukocyte numbers lagged behind that of mucosal mast cells. The results supported the concept of CD4+ T cell help in the abomasal mucosa and defined the sequential expression of components of the immunological responses potentially mediating resistance to T. axei. In sheep No. 701, persistence of adult worms was associated with lower mucosal IgE+ cell and eosinophil responses compared with the other dosed sheep.

Production and characterisation of monoclonal antibodies recognising ovine IgE.

Two monoclonal antibodies (mAbs), XB6 and YD3, which recognise ovine immunoglobulin E (IgE) were produced. Mast cells isolated from ovine intestinal mucosa were used as a source of IgE to immunize mice. Culture supernatants of hybridomas were screened by immunoassays on small-intestine tissue sections, isolated mucosal cells, and dot blots of lysed mast cell homogenate. Two mAbs were chosen for their specific binding to mast cells. Antigen bound by these mAbs was purified by immunoaffinity chromatography using XB6 mAb, and this produced two bands consistent with IgE heavy chain (86,000 Daltons) and immunoglobulin light chain (28,000 Daltons) when run under reducing conditions on SDS-PAGE gels. Purified IgE was shown on dot blots to react weakly with mAb to chimeric ovine IgE and strongly to polyclonal anti-sheep antibodies. The two mAbs induced an immediate hypersensitivity-like reaction when injected into the skin of sheep. The mAbs bound to mast cells and other mononuclear cells, presumably IgE-secreting B-cells in mesenteric lymph node sections. These mAbs proved useful for detecting IgE-bearing cells in various ovine tissues, for purifying mast cells from cell isolates by panning and immunomagnetic bead separation, for purifying serum IgE using immunoaffinity chromatography and for detecting IgE in an ELISA. Competitive binding assays showed that the two mAbs bind to different epitopes on IgE. These mAbs will be useful in research applications and in diagnostic assays.

Immune responsiveness of nematode-resistant or susceptible Romney line-bred sheep to continuous infection with Trichostrongylus axei.

Two divergent lines of Romney sheep have been selected on the basis of differences in faecal egg counts (FEC) to natural poly-generic parasite challenge in New Zealand. However, it is not known if the expression of resistance or susceptibility extends to parasitic nematodes that were not a major part of the selection challenge. To examine this, the immune response to infection with Trichostrongylus axei was examined in these sheep lines. Changes in the proportions of CD5+, CD4+, CD8+, T19+ and CD45R+ lymphocytes and parasite specific antibody titres in peripheral blood were monitored each week in six resistant and six susceptible line lambs that were maintained indoors in pens during the course of 14 weekly infections with 10,000 T. axei larvae. No difference in FEC was observed. Similarly, no significant difference in T. axei specific antibody titre between sheep lines was seen although antibody titre increased steadily from Week 4. Significant increases in the proportions of CD5+, CD4+, CD8+ and T19+ cells occurred in both resistant and susceptible line lambs during Weeks 1-4 of infection. Following peak levels, proportions of CD5+, CD4+ and CD8+ cells fell with the rate of decline of CD5+ and CD4+ cells significantly greater in the resistant line lambs. Proportions of CD45R+ cells showed significant changes with time that were the inverse of those of CD5+, CD4+ and CD8+ cells. Susceptible line lambs showed higher proportions of CD5+ and lower proportions of CD45R+ cells than resistant lambs before infection with T. axei. Overall, during infection, these differences were maintained while CD4+ and T19+ levels were higher in the susceptible line lambs.

The immune response of sheep surgically modified with intestinal loops to challenge with Trichostrongylus colubriformis.

Aspects of the local immune response to nematode challenge were investigated in vivo in isolated loops of the upper small intestine of mature sheep that were immunised by repeated infections with Trichostrongylus colubriformis infective larvae (L3). Groups of 3 sheep were challenged either through the loop (Group 1) or orally (Group 2) with T. colubriformis L3, the third group served as unchallenged controls (Group 3). Nematode specific antibody levels, mast cell proteinase levels (SMCP) and larval migration inhibition (LMI) activity were determined in loop secretions for 4 weeks after challenge. The intestinal loops remained functional throughout the experiment. Groups 1 and 2 were re-challenged 2 weeks after the first challenge, and all 3 Groups were slaughtered 2 weeks later. Histopathological examination showed elevated numbers of globule leukocytes (GL) in both the nematode-challenged loop and unchallenged small intestine of Group 1 and small intestine of Group 2 indicating that nematode infections induce the local appearance of large numbers of GL. Oral, but not loop challenge caused increased antibody levels in loop secretions when compared to unchallenged controls. Only loop-challenged sheep showed a peak in loop fluid SMCP levels 10-13 days after the first challenge which coincided with a peak in numbers of mucosal GL. The isolated loops of all 3 groups showed highly elevated numbers of eosinophils when compared to the intact small intestine. Loop fluid of all 3 groups showed a high level of LMI activity reflecting the high level of nematode-resistance induced by the immunisation procedures. Sheep in Groups 1 and 2 were both able to expel challenge infections, and when compared to Group 3, showed higher blastogenic activity of unstimulated cells derived from a mesenteric lymph node in the region of the challenged part of the intestine. The present experiment showed that surgically constructed intestinal loops provided a model system by which the substantial changes associated with the local intestinal immune response to challenge with T. colubriformis could be investigated.

Antibody responses of grazing alpacas (Lama pacos) in New Zealand to intestinal nematodes.

Alpaca (Lama pacos) were grazed for 10 months (October 1992-June 1993) on pasture with sheep or on pasture which had been recently grazed by sheep. The alpaca, of various age groups, totalled 94 at the beginning of the experiment and during the course of the experiment 32 progeny (cria) were born, 10 in spring 1992 and 22 in autumn 1993. Serum levels of specific antibodies to excretory/secretory antigens of the third larval stage (L3) of Cooperia curticei, Ostertagia circumcincta or Trichostrongylus colubriformis and somatic antigens from adult T. colubriformis were determined at monthly intervals by ELISA. Faecal egg count and live-weight were determined monthly and fleece-weight was measured at shearing. Three days after the birth of the cria, serum antibody levels ranged from 0.46-0.85 optical density units for the L3 antigens and averaged 0.22 for the adult T. colubriformis antigen. These levels declined to 0.1-0.24 and 0.06 respectively by 2-3 months of age. Subsequently, antibody levels increased steadily to reach maximal adult levels at approximately 23-26 months. Antibody levels were negatively correlated with FEC, but positively correlated with live-weight at 7 months although at 15 months antibodies and live-weight were negatively correlated. A positive correlation was found between weight and FEC. Fleece-weight showed no correlation with antibody level, a positive correlation with weight and a negative correlation with FEC. The relationships among antibody responses, FEC, live-weight and fleece-weight observed for alpaca in this experiment suggest that antibody responses might provide a useful indicator of alpaca immuno-responsiveness and has potential for use as a parameter for selection of alpaca with reduced FEC.

Supravital staining of eosinophils.

Live eosinophils when mixed with Acridine Orange solution and viewed microscopically using u.v. light show very intense colours of their granules (yellow, orange and red) and green nuclear staining. Their active movement, translocation of granules and degranulation can be observed in vitro. Using this method, live eosinophils can be easily differentiated and enumerated.

Nematode burdens and immunological responses following natural challenge in Romney lambs selectively bred for low or high faecal worm egg count.

Breeding lines of Romney sheep, selected as lambs for consistently low or high faecal nematode egg count (FEC) following periods of natural challenge, have been maintained at Wallaceville for some years. In order to determine the extent to which FECs in low and high genotypes reflected their ability to resist the establishment of gastro-intestinal nematode burdens, we investigated the infection status and immune responses in 8- to 9-month-old progeny of selected rams from low and high FEC breeding lines following a period of grazing without anthelmintic treatment in autumn/early winter. In each of the 2 years of the study, outcross male progeny of the two lowest FEC (LFEC) (i.e. most 'resistant') and two highest FEC (HFEC) (i.e. most 'susceptible') rams from the divergent lines were slaughtered shortly after autumn/early winter FECs had been analysed. Post-mortem worm counts and examination of intestinal histology were then undertaken. Blood samples collected before slaughter in the second year of the study were assayed to measure serum levels of Trichostrongylus colubriformis-specific antibody and immunoglobulins (IgG1 and IgM), and numbers of circulating eosinophils. Overall, correlations between pre-slaughter FEC and total trichostrongyle burdens in the lambs proved to be very high (0.91 and 0.85, respectively, for the 2 years studied). In the first year, LFEC lambs, which were shedding only 28.6% as many strongyle eggs as their HFEC counterparts at slaughter, were found to harbour 37.6% as many adult trichostrongyle worms, while in the second year, LFEC lambs, which were shedding 16.1% as many strongyle eggs as their HFEC counterparts at slaughter, were found to harbour 33.5% as many adult trichostrongyle worms. Results, particularly in the second year, confirmed that significantly fewer worms of most of the important abomasal and small intestinal nematode species which infest lambs in New Zealand (i.e. Haemonchus contortus, Ostertagia circumcincta, Cooperia curticei, Nematodirus spathiger, T. colubriformis, and Trichostrongylus vitrinus) had established in the LFEC genotypes than in their HFEC counterparts. In addition, in utero egg counts of female intestinal Trichostrongylus spp. were significantly lower in LFEC lambs than in their HFEC counterparts, indicating a reduction in fecundity of those worms which did establish. There was also some evidence of an effect of host response on the developmental composition of burdens in the case of some worm species. In relation to host responses, numbers of globule leucocytes/mucosal mast cells in the intestinal mucosa were significantly higher (P < 0.01) in LFEC lambs than in HFEC lambs in both years of the study. Numbers of connective tissue type mast cells and eosinophils in the intestinal mucosa were also significantly higher in LFEC lambs but only in the second year of the study (P < 0.01 and P < 0.05, respectively). Numbers of circulating eosinophils did not differ significantly between the genotypes. T. colubriformis-specific antibodies, IgG1 and IgM to both L3 and adult worm antigens were all significantly higher (P < 0.01 or P < 0.05) in LFEC lambs than in HFEC lambs.

Secretion of anti-parasite substances and leukotrienes from ovine gastrointestinal tissues and isolated mucosal mast cells.

The presence of larval migration inhibitory (LMI) compounds in the gastrointestinal mucus of nematode resistant sheep has been shown previously to be associated with increased numbers of gastrointestinal mucus of nematode resistant sheep has been shown previously to be associated with increased numbers of gastrointestinal mucosal mast cells (MMC) and globule leukocytes (GL). This experiment was designed to determine if LMI compounds were secreted by MMC/GL in response to nematode antigenic challenge and if so, could secretion account for levels observed in mucus. Romney sheep were immunized by repeated cycles of infection with Trichostrongylus colubriformis or Haemonchus contortus larvae and anthelmintic treatment. After slaughter, gastrointestinal tissue was taken for examination of histology and mucus anti-parasite activity. Segments of small intestine were ligatured to form sacs which were incubated with exsheathed nematode larvae or larval excretory/secretory antigens. Tissue slices from small intestine or abomasum were also incubated with nematode larvae or antigens. After homologous challenge, levels of leukotrienes secreted into small intestinal tissue sacs were significantly higher than levels in heterologously challenged sacs or unimmunized sheep intestinal sacs challenged with larvae of any nematode species (279.4 +/- 33.7, 141.0 +/- 27.8 and 39.5 +/- 15.2 ng h-1 respectively). Tissue slices gave a similar pattern of leukotriene secretion. LMI activity was also significantly elevated in intestinal sacs from immunized sheep challenged homologously with nematode larvae or antigen (64 +/- 10 and 68 +/- 14% respectively cf. heterologous challenge 32 +/- 10% and unimmunized sheep sacs 15 +/- 6%). Histological examination of abomasal and small intestinal sections showed that immunized sheep had significantly greater numbers of MMC/GL than unimmunized sheep. MMC/GL isolated and purified from immunized sheep secreted leukotrienes and compounds having LMI activity when cultured with homologous nematode larvae or antigens. Secretion of leukotrienes and molecules having LMI activity from MMC/GL could account for the levels of these substances observed in small intestine mucus.

Serum mast cell proteinase responses of sheep to challenge with Trichostrongylus colubriformis and the effect of dexamethasone treatment.

Eight-month-old random bred Romney wethered lambs were reared nematode-free in pens and assigned to 4 groups of 5 lambs. Lambs in 2 groups were dosed orally, twice a week, with 5000 Trichostrongylus colubriformis infective larvae (L3) for the duration of the experiment. These 2 groups were treated weekly with dexamethasone (0.5 mg kg(-1) body-weight), one between days -7 and 70, the other between days 77 and 147. A third group was dosed with L3 until anthelmintic treatment on day 133. A fourth group remained uninfected throughout and served as a control group. Nematode eggs in sheep faeces (FEC) were monitored at weekly intervals. Serum samples were taken twice a week and assayed for sheep mast cell proteinase (SMCP). Serum levels of SMCP in uninfected control sheep were 459 +/- 190 pg ml(-1). Twenty-eight days after nematode dosing commenced, SMCP levels were significantly above control sheep levels and after 49 days reached a plateau level of 1154 +/- 364 pg ml(-1). The SMCP response persisted even after cessation of dosing, and SMCP levels remained significantly above control levels to the end of the experiment (day 213). Dexamethasone treatment prevented elevation of SMCP and resulted in a rapid reduction of extent SMCP levels in resistant sheep. Overall, serum levels of SMCP were significantly correlated (P<0.001) with specific anti-T. colubriformis L3 antibody in serum (r = 0.601, d.f. = 78), blood eosinophils (r = 0.609, d.f. = 78) and log(FEC+15) (r = -0.521, d.f. = 78). These results show that serum levels of SMCP correlate with other indicators of parasitism and may have potential use as a non-invasive indicator of gastrointestinal mast cell responses to nematode infection.

Genetic factors affecting antibody responses to four species of nematode parasite in Romney ewe lambs.

Serum levels of antibodies (Ab) and immunoglobulin G1 (IgG1) to the larval (L3) stage of the internal parasites Cooperia curticei and Trichostrongylus colubriformis and levels of Ab to the L3 stages of Haemonchus contortus and Ostertagia circumcincta were determined in 1432 Romney ewe lambs which were born on one farm in 1990 and 1991 and were the progeny of 63 rams. The objectives were to estimate heritabilities of, and genetic correlations among, the serum concentrations in newly weaned lambs under commercial conditions and to estimate genetic correlations of Ab and IgG1 with production traits. Lambs were exposed to a natural parasite challenge on pasture, following an anthelmintic drench at weaning. Blood and faecal samples from 4- to 6-month-old lambs were then taken when the mean faecal nematode egg count of a monitor group reached 800-1500 eggs g-1. Heritabilities for the serum levels of the four Abs ranged from 0.25 +/- 0.05 to 0.37 +/- 0.06. Heritabilities for the level of IgG1 developed against C. curticei was 0.19 +/- 0.04 and against T. colubriformis, 0.18 +/- 0.05. Genetic correlations between Abs for the 4 species were high, averaging 0.84, and between the two IgG1s it was 0.82. The genetic correlations between Ab or IgG1 levels and weight or gain traits were negative (for the 6 significant values out of 18), with yearling fleece weight positive (for the 2 significant values out of 6), whilst those with loge faecal egg count were all negative (average -0.15).(ABSTRACT TRUNCATED AT 250 WORDS)

Variation in immune responsiveness of sheep to the antigens of intestinal nematodes and blowfly larvae.

The total and IgG1 antibody responses to the intestinal nematode parasites Haemonchus contortus and Trichostrongylus colubriformis were measured in the serum of 160 lambs, 4 months of age. These antibodies had developed as the result of natural exposure to the parasites on pasture. Three sires were examined and strong sire effects on half-sib progeny were found. Plotting of ELISA antibody results in two dimensions revealed clustering of responses within sire groups. Bimodal antibody distributions were also observed within sire groups and the whole population for T. colubriformis. A bimodal distribution of antibodies to H. contortus was found for one sire group but not for the whole population. The injection of blowfly larvae (Lucilia cuprina) extract into 42/160 lambs at a later age (12 months) was followed by increased antibodies to L. cuprina and an apparent increase in antibodies to T. colubriformis. A bimodal distribution for antibodies to L. cuprina was found in one sire group and in the whole population. These bimodal distributions of antibodies to L. cuprina did not coincide with the distribution of antibodies to T. colubriformis or H. contortus, measured on the same serum samples. It was concluded that high and low responder sire groups could be differentiated in lamb populations for all three parasites. These effects persisted during lamb maturation and appeared to be genetic effects. Finally, cross-reacting antibodies between L. cuprina and T. colubriformis appear to be stimulated by injection of L. cuprina antigens.

Immunization of sheep against parasitic nematodes leads to elevated levels of globule leukocytes in the small intestine lumen.

In sheep that had been given three immunizing infections with Trichostrongylus colubriformis and Ostertagia circumcincta infective (L3) larvae, drenched after the last infection and challenged with larvae of the same species, there was a significant increase in numbers of small intestine mucosal tissue globule leukocytes (TGLs) and lumenal globule leukocytes (LuGLs) compared with sheep that had only been drenched and challenged. There was a positive correlation between the numbers of LuGLs and TGLs in the small intestine but the ratio of these two cell types was lower in non-immunized than immunized sheep. In immunized sheep positive correlations were observed between LuGLs and levels of arylsulphatase and peroxidase in the intestinal mucus and between arylsulphatase and larval migration inhibition (LMI) activity in mucus. Lumen eosinophils correlated with blood eosinophils, serum antibody against T. colubriformis correlated with peroxidase in the mucus and blood eosinophils correlated with nematode specific IgM levels in the intestinal mucus. In the abomasum, TGLs were present but not LuGLs. Sheep repeatedly infected with T. axei also had significantly more LuGLs in the small intestine than control animals. Two sheep that had a surgically prepared isolated small intestinal loop, after oral infection with T. colubriformis had TGLs and LuGLs in the intact intestine, but not in the isolated loop. Significantly more LuGLs were produced in sheep by allowing repeated T. colubriformis L3 infections to develop to adult stages compared to sheep treated with the same number of larvae, but where the infections were terminated by drenching at various intervals.

Antibody responses of sheep to challenge with Trichostrongylus colubriformis and the effect of dexamethasone treatment.

Eight-month-old random bred Romney wether lambs were reared nematode-free in pens and assigned to 4 groups each of 5 lambs. Lambs in 3 groups were infected orally, twice a week, with 5000 Trichostrongylus colubriformis infective larvae (L3), a control group remained unifected throughout. Two infected groups were treated with dexamethasone (0.5 mg kg-1 bodyweight), one between days -7 and 77, the other between days 77-154. Nematode challenge infection was withheld from the third group from day 133 after anthelmintic treatment. Nematode eggs in sheep faeces (FEC) were monitored at weekly intervals. T. colubriformis-specific antibody levels were determined twice a week and specific immunoglobulin isotypes (IgA, IgG1, IgG2 and IgM) determined weekly in serum samples using ELISA. Resistance, as measured by FEC, was expressed by 35 days after L3 infection began but sheep dosed with dexamethasone did not develop resistance. Extant resistance was abrogated in sheep dosed with dexamethasone. Nematode challenge resulted in elevated serum levels of antibodies to T. colubriformis L3 excretory/secretory antigens, these consisted predominantly of IgG1 and IgM. The IgG1 response was more persistent than the IgM response. Specific serum IgA and IgG2 responses were low, but significant, in nematode-challenge sheep. Dexamethasone treatment prevented the antibody responses and resulted in a rapid reduction of extant antibody levels in resistant sheep. Weight gain was reduced by nematode challenge with or without dexamethasone treatment compared with control sheep.

Improved bioassay for estimation of inhibitory effects of ovine gastrointestinal mucus and anthelmintics on nematode larval migration.

A bioassay based on the ability of substances present in ovine gastrointestinal mucus or anthelmintics to paralyse third stage larval (L3) nematodes and inhibit their passage through 20 microns nylon mesh sieves [larval migration inhibition (LMI) activity] is described. Factors influencing the reproducibility of the bioassay were examined using exsheathed L3 of the sheep gastrointestinal nematode parasite Trichostrongylus colubriformis. Levamisole, morantel tartrate and piperazine were shown to inhibit L3 migration in the bioassay. The bioassay was used to demonstrate that gastrointestinal mucus from nematode-resistant sheep possessed greater L3 inhibitory activity than mucus from nematode-susceptible sheep. LMI activity of mucus used in this bioassay was significantly correlated with LMI activities obtained using two previously described similar bioassays. The action of mucus components on nematode larvae was shown to be reversible in the bioassay. The modified assay has advantages over other bioassays as it avoided the use of temperature-dependent agar blocks, reduced the number of L3 required to a more manageable size, and the whole experiment could be performed on a 48-well culture plate. The reproducibility, high correlation with other bioassays, ease of performance, suitability for testing a large number of samples and low cost make this modified assay the method of choice for determining antiparasitic activity of gastrointestinal mucus components and as a screen for potential anthelmintics.

The effects of anthelmintics on ovine larval nematode parasite migration in vitro.

The anthelmintics ivermectin, levamisole, morantel tartrate and thiabendazole all inhibited, in vitro, the motility of third stage larvae (L3) of Trichostrongylus colubriformis. The bioassay, based on the inhibition of L3 migration from agar gels, yielded sigmoid dose-response curves for ivermectin, levamisole and morantel tartrate, but not thiabendazole. The concentration of levamisole giving 50% inhibition of migration (EC50) was determined for Cooperia curticei, Haemonchus contortus, Nematodirus spathiger, Ostertagia circumcincta, Trichostrongylus axei, T. colubriformis and T. vitrinus. EC50s differed between species but within species the EC50s for ensheathed and exsheathed L3 were similar except for N. spathiger which showed significantly higher EC50 for the ensheathed L3. No difference between EC50s for levamisole-resistant and susceptible strains of T. colubriformis were found. Similarly, morantel-resistant and susceptible strains of T. colubriformis could not be differentiated in this bioassay. The inhibition of L3 motility by known anthelmintic compounds in this bioassay suggests that the bioassay could be used as a screen for potential new anthelmintics.

Sheep small intestinal mast cells/globule leucocytes: ability to divide.

The number of mucosal mast cells/globule leukocytes (MMC/GLs) increase in the intestinal mucosa in response to nematode parasite infections but it is not known if this accumulation is due to in situ cell division, derivation from elsewhere or some combination of both. To determine if MMC/GLs can divide, cells were obtained from immunized Romney sheep and cultured in vitro in RPMI 1640. For cultures supplemented with 10, 20 or 30% foetal lamb serum (FLS) or foetal calf serum (FCS) and without concanavalin A (Con A), cell division had ceased by day 2, but with Con A (3 micrograms ml-1) cell division continued to day 9. Better growth of cells was obtained with the higher concentrations of serum. However the use of 30% or 50% autologous serum with Con A lead to cell death but 10% serum permitted limited growth. The detrimental effect of autologous serum could be overcome by increasing the Con A concentration. It was established that an alpha-macroglobulin present in autologous serum can bind Con A. This macroglobulin appears to have a higher avidity for Con A than does the receptor(s) on the surface of MMC/GLs. Our data seem to indicate that a direct interaction of Con A with the cell surface or a receptor(s) is responsible for MMC/GLs division.